Microbial utilization and degradation of alkanes was discovered almost a century ago. Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates. The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenases (BMO) are specialized on gaseous small-chain alkanes (C1 to C4), while medium-chain (C5 to C16) alkane hydroxylation appears to be the domain of the CYP153A6 and AlkB enzyme families.
Conversion of C1 to C4 alkanes to the corresponding alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. (van Beilen, J. B., et. al., 2007, Appl. Microbiol. Biotechnol., 74, 13-21). Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth.
CYP153 is a family of enzymes that has recently been shown to hydroxylate alkanes to the corresponding alkanols. (Maier, T. H., et. al., 2001, Biochem. Biophys. Res. Commun., 286, 652-658; Funhoff, E. G., et. al., 2006, J. Bacteriol., 188, 5220-5227; Funhoff, E. G., et. al., 2007, Enzyme Microb. Technol., 40, 806-812; and, van Beilen, J. B., et. al. 2006, Appl. Environ. Microb., 72, 59-65). (16, 9, 10, 31). This family of heme-containing cytochrome P450 monooxygenases has been the subject of biochemical studies and known substrates includes alkanes containing five to eleven carbons. (Müller, R., et. al., 1989, Biomed. Biochim. Acta, 48, 243-254; Maier, T. H., et. al., 2001, Biochem. Biophys. Res. Commun., 286, 652-658; and, Funhoff, E. G., et. al., 2006, J. Bacteriol., 188, 5220-5227). (9,16,19). The best characterized member of this family, CYP153A6, hydroxylates it's preferred substrate, octane, predominantly at the terminal position. (Müller, R., et. al., 1989, Biomed. Biochim. Acta, 48, 243-254; Funhoff, E. G., et. al., 2007, Enzyme Microb. Technol., 40, 806-812; and, Kubota, M., et. al., 2005, Biosci. Biotechnol. Biochem., 69, 2421-2430). (9,10,14). Nucleotide and amino acid sequences for CYP153A6 from Myobacterium sp. HXN-1500 can be found in, and are hereby incorporated by reference from, the GenBank database under the accession Nos. AJ783967 (SEQ ID NO: 1) and Q65A64 (SEQ ID NO: 2), respectively. However, the CYP153A6 gene used as a parent gene in this study was found to lack the first three codons compared to the AJ783967 sequence, probably due to cloning procedures. However, the encoded protein was otherwise identical, fully functional and performed identical to the Q65A64 protein under all analyzed conditions. Therefore, it will also be referred to as an amino acid having the sequence set forth in SEQ ID NO: 3.
The soluble class II-type three-component CYP153A6 enzymes are the main actors in medium-chain length alkane hydroxylation by the cultivated bacteria to date. (van Beilen, J. B., et. al. 2006, Appl. Environ. Microb., 72, 59-65). Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101, a camphor hydroxylase) and P450 BM3 (CYP102A, a fatty acid hydroxylase) (Fasan, R., et. al., 2007, Angew. Chem. Int. Ed. Engl., 46, 8414-8418; Xu, F. et. al., 2005, Angew. Chem. Int. Ed., 44, 4029-4032). However, the resulting enzymes hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions. Highly selective and desirable terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase. (Peters, M., et. al., 2003, J. Am. Chem. Soc., 125, 13442-13450).
Previous approaches, some of which are described above, relate to in vitro evolution of hydroxylase enzymes that does not provide the opportunity to screen for improved activity on a specific alkane substrate directly. Further, the prior methods lead to low or no terminal hydroxylation activity and often result in high uncoupling.